1. Field of the Invention
This invention relates to coat protein (CP) genes derived from WT strains of cucumber mosaic virus (CMV). More specifically, the invention relates to the genetic engineering of plants and to a method for conferring viral resistance to a plant using an expression cassette encoding CP genes of WT strains of CMV.
2. Description of the Prior Art
Many agriculturally important crops are susceptible to infection by plant viruses, particularly CMV, which can seriously damage a crop, reduce its economic value to the grower, and increase its cost to the consumer. Attempts to control or prevent infection of a crop by a plant virus such as CMV have been made, yet viral pathogens continue to be a significant problem in agriculture.
Scientists have recently developed means to produce virus resistant plants using genetic engineering techniques. Such an approach is advantageous in that the genetic material which provides the protection is incorporated into the genome of the plant itself and can be passed on to its progeny. A host plant is resistant if it possesses the ability to suppress or retard the multiplication of a virus, or the development of pathogenic symptoms. xe2x80x9cResistantxe2x80x9d is the opposite of xe2x80x9csusceptible,xe2x80x9d and may be divided into: (1) high, (2) moderate, or (3) low resistance, depending upon its effectiveness. Essentially, a resistant plant shows reduced or no symptom expression, and virus multiplication within it is reduced or negligible. Several different types of host resistance to viruses are recognized. The host may be resistant to: (1) establishment of infection, (2) virus multiplication, or (3) viral movement.
CMV is a single-stranded (+) ribonucleic acid (RNA) plant virus that has a functionally divided genome. The virus genome contains four RNA species designated RNAs 1-4. RNAs 3 and 4 encode the coat protein (CP) which is a protein that surrounds the viral RNA and protects the viral RNA from being degraded. Only RNAs 1-3 are required for infectivity because the CP, which is encoded by RNA 4, is also encoded by RNA 3.
Several strains of CMV have been classified using serology, host range, peptide mapping, nucleic acid hybridization, and sequencing analyses. These CMV strains can be divided into two groups, which are designated xe2x80x9cWTxe2x80x9d (also known as subgroup I) and xe2x80x9cSxe2x80x9d (also known as subgroup II). The S group consists of at least three members. The WT group is known to contain at least 17 members.
Expression of the CP genes from tobacco mosaic virus, alfalfa mosaic virus, CMV, and potato virus X, among others, in transgenic plants has resulted in plants which are resistant to infection by the respective virus. Heterologous protection can also occur. For example, the expression of CP genes from watermelon mosaic virus-2 (WMV-2) or zucchini yellow mosaic virus (ZYMV) in transgenic tobacco plants has been shown to confer protection against six other potyviruses: bean yellow mosaic virus, potato virus Y, pea mosaic virus, clover yellow vein virus, pepper mottle virus, and tobacco etch virus. However, expression of a preselected CP gene does not reliably confer heterologous protection to a plant. For example, transgenic squash plants containing the CMV-C CP gene, a WT virus, which have been shown to be resistant to the CMV-C strain are not protected to the same degree against several other, highly virulent WT strains of CMV.
Thus, a need exists for plants resistant to WT strains of CMV.
This invention provides: an isolated and purified deoxyribonucleic acid (DNA) molecule that encodes the CP for the V27 strain of CMV (CMV-V27), and a chimeric expression cassette comprising this DNA molecule; an isolated and purified DNA molecule that encodes the CP for the V33 strain of CMV (CMV-V33), and a chimeric expression cassette comprising this DNA molecule; and an isolated and purified DNA molecule that encodes the CP for the V34 strain of CMV (CMV-V34), and a chimeric expression cassette comprising this DNA molecule; and an isolated and purified DNA molecule that encodes the CP for the A35 strain of CMV (CMV-A35), and a chimeric expression cassette comprising the DNA molecule. Another embodiment of the invention is exemplified by the insertion of multiple virus gene expression cassettes into one purified DNA molecule, e.g., a plasmid. Each of these cassettes also includes a promoter which functions in plant cells to cause the production of an RNA molecule, and at least one polyadenylation signal comprising 3xe2x80x2 nontranslated DNA which functions in plant cells to cause the termination of transcription and the addition of polyadenylated ribonucleotides to the 3xe2x80x2 end of the transcribed messenger RNA (mRNA) sequences, wherein the promoter is operably linked to the DNA molecule, and the DNA molecule is operably linked to the polyadenylation signal. Preferably, these cassettes include the promoter of the 35S gene of cauliflower mosaic virus (CaMV-355 gene) and the polyadenylation signal of the CaMV-35S gene (CaMV-35S).
Also provided are bacterial cells, and transformed plant cells, containing the chimeric expression cassettes comprising the CP genes derived from the CMV-V27, CMV-V33, CMV-V34, or CMV-A35 strains, and preferably the 35S promoter and the polyadenylation signal of the CaMV-35S gene. Plants are also provided, wherein the plants comprise a plurality of transformed cells containing the chimeric CP gene expression cassettes derived from the CMV-V27, CMV-V33, CMV-V34, or CMV-A35 stains, and preferably the promoter and the polyadenylation signal of the CaMV gene. Transformed plants of this invention include tobacco, beets, corn, cucumber, peppers, potatoes, melons, soybean, squash, and tomatoes. Especially preferred are members of the Cucurbitaceae (e.g., squash and cucumber,) and Solanaceae (e.g., peppers and tomatoes) family.
Another aspect of the present invention is a method of preparing a CMV-resistant plant, such as a dicot, comprising: transforming plant cells with a chimeric expression cassette comprising a promoter functional in plant cells operably linked to a DNA molecule that encodes a CP of a WT strain of CMV, e.g., V27, V33, V34, or A35; regenerating the plant cells to provide a differentiated plant; and identifying a transformed plant that expresses the CMV CP at a level sufficient to render the plant resistant to infection by the specific strains of CMV disclosed herein.
As used herein, with respect to a DNA molecule or xe2x80x9cgene,xe2x80x9d the phrase xe2x80x9cisolated and purifiedxe2x80x9d is defined to mean that the molecule is either extracted from its context in the viral genome by chemical means and purified and/or modified to the extent that it can be introduced into the present vectors in the appropriate orientation, i.e., sense or antisense. As used herein, the term xe2x80x9cchimericxe2x80x9d refers to the linkage of two or more DNA molecules which are derived from different sources, strains or species (e.g., from bacteria and plants), or the linkage of two or more DNA molecules, which are derived from the same species and which are linked in a way that does not occur in the native genome. As used herein, xe2x80x9cexpressionxe2x80x9d is defined to mean transcription or transcription followed by translation of a particular DNA molecule.